Extraction of Ascorbic Acid from Plant Tissues WARD B. DAVIS Agricultural Chemical Research Division, Bureau of Agricultural Chemistry and Engineering, U. S. Department of Agriculture, Los Angeles, Calif.
H E extraction of ascorbic acid by a new procedure which utilizes the Waring Blendor (2) is briefly described. This procedure will avoid the laborious grinding necessary for plant tissue hard to triturate, as in the method of Thornton (6) and others. Plant material was extracted by covering the cutting knives of the Blendor with about 150 ml. of extractant, adding a sample of 10 to 20 grams, and running the motor a t high speed for 5 minutes. The Blendor and reagents were kept in a room maintained near 5" C. Sufficient Super-Cel filter aid was added to the finely divided material suspended in the extractant t o give quick filtration by suction on a Biichner funnel. Convenient aliquots of the clear, almost colorless filtrate which was made t o a volume of 150 ml. were titrated with 2,6-dichlorophenolindophenol. The dye was standardized by the method of Menaker and
T
TABLE I. COMPARISON OF METHODSFOR EXTRACTION OF AsCORBIC ACID (MQ. ASCORB~C ACID PER GRAMFRESHWEIQHT) -Hand a Parsley First extract Second extract Third extract Residue Total First ext 7 of total Extract& &e, min. Cabbage First extract Second extract Third extract Residue Total First ext., % of total Extraction time, min. Frozen peas First extract Second extract Third extract Residue Total First ext %, of total Extractizn time, min.
Methodb
1.93 0.07 0.01 0.07
2.20 0.06 0.02 0.12
-Blendor a
Methodb
2.00 0.03 0.01 0.02 2.06 97.09 10
2.20 0.03 0.01 0.02 2.26 97.38 10
- - - 2.08
2.40
92.78 24
91.67
0.32 0.01 0.00
0.30 0.01 0.00
0.30 0.01 0.01
0.23 0.01 0.01
0.32 93.76 19
0.33 90.90 12
0.26 88.47 13
20
0.01
0.01
0.34 94.12 19
0.22 0.01
0.19
0.01
::ti - -
;:"0
0.22 86.36 18
0.25 88.00 18
0.22 86.30 10
0.23 78.80 11
Chard First extract First ext., % of total Mustard greens First extract First ext., 70of total
0.40 75.54
0.42 79.83
1.11 90.16
1.07 85.88
Potato First extract First ext., of total
0.09 86.79
0.07 88.49
1.75
1.92
Grapefruit peela Av. of 4 samples
Fresh peas5 Av. of 6 samples 0.07 Av. of 6 samples, acid-washed Super-Cel Av. of 6 samples, centrifuged a Extractant, 2% metaphosphoric acid.
... ...
0.11 0.16 0.16
Guerrant (4). Except where 2 per cent metaphosphoric acid was used, the extractant was a mixture of 3 per cent trichloroacetic acid and 2 per cent metaphosphoric acid. After extraction, 8 per cent acetic acid was used for diluting the mixture to volume. Ballentine's iodate method (1) was used on some of the tissues. The end point is sharper and results are higher than by the dye titration. The Blendor method of extracting ascorbic acid was compared with the usual method of grinding by hand. Five grams of the plant tissue were ground 5 minutes with 25 ml. of extractant and 3 ml. of sand to a thin paste, which wm poured into a 50-ml. conical centrifuge tube. Samples ground in duplicate were centrifuged a t the same time for 3 minutes. The fairly clear liquid was decanted, and then 10 ml. of fresh extractant, used to rinse the mortar, were stirred with the residue in each tube. These were centrifuged and decanted, and a final extraction was made with 5 ml. The total liquid decanted was made to 50 ml. This was the first extract. An aliquot of the first extract was made to about 50 ml. and titrated. Two additional extractions were made by grinding the residue in the mortar again for 2 minutes, each time with 25 ml. of extractant, and centrifuging. The residue of tissue fragments and sand was diluted to 50 or 100 ml. and titrated. The end point, ordinarily obscured by suspended particles, was clearly seen after centrifuging a small quantity of the suspension in an angle centrifuge for 1 minute. Twenty-gram samples of the same tissues extracted by the hand grinding were reduced in the Blendor, in the manner already described, for 5 minutes. Two additional extractions of 2 minutes each were made on each sample in duplicate, Only 5 grams of grapefruit peel and 10 grams of fresh peas were used in the Blendor. The extractant for these two tissues was 2 per cent metaphosphoric acid (3). Samples of the fresh peas from the same lot were weighed, frozen, and kept frozen until extracted to avoid loss of ascorbic acid on standing. Centrifuging the fresh pea tissue after extraction gave the same results as filtration with acid-washed filter aid. Some tissues foam in the Blendor, making the transfer to the centrifuge tubes bothersome. A few drops of alcohol aid in causing the foam t o subside. As shown in Table I, the new procedure appears not only to be just as efficient but also to require less time and effort on the part of the worker than the older, grinding method. Even in cabbage tissue, which contains ascorbic acid oxidase, the new procedure seems satisfactory. The titration figures for the tissue residues, although only approximate in nature, indicates the completeness of the extractions. Higher results were obtained from samples in which acidwashed in comparison with unwashed Super-Cel was used. To test the recovery of added ascorbic acid, 20 mg. of pure ascorbic acid were added to the titrated residues from 20 217
INDUSTRIAL AND ENGINEERING CHEMISTRY
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grams of extracted tissues mixed with filter aid and fragments of filter paper. After the ascorbic acid was stirred into the residue, the extractant was added and the suspension was placed in the Blendor. The procedure was then the same as described for the extraction of fresh material. The degree of completeness of extraction of added ascorbic acid was similar t o that of the first extraction of naturally occurring ascorbic acid. The total percentage recovered varied from 90 to 101 per cent. Boiled cabbage tissue residue gave about the same percentage recovery of added ascorbic acid as the unboiled residue. A recovery of 99 per cent was made when ascorbic acid was added t o t h e extractant containing
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no plant tissue and the motor was run a t high speed for 10 minutes. Much time and effort may be saved by this procedure, because a large number of workers have been making the ascorbic acid assay by the chemical method.
Literature Cited (1) Ballentine, R.8 1x0. ENG.CHEM.9 ANAL.ED.,13, 89 (1941). (2) Davis, W. B.,IND.ENG.CHEM.,NEWSED., 17, 752 (1939). (3) King, C. G., IND.ENG.CHEX.,ANAL.ED., 13, 228 (1941). (4) Menaker, M. H., and Guerrant, N. B., Ibid., 10, 25 (1938). ( 5 ) Thornton, N. C.,Contrib. Boyce Thompson Inst , 9,273 (1938). CovTRiBuTroN 37 f r o m t h e Division of Agricultural
Chomioal Research.
Effect of Reinforcing Pigments on Rubber Hydrocarbon F. S. THORNHILL AND W. R. SMITH Godfrey L. Cabot, Iiic., Boston, Mass.
The unsaturation values and amount of combined sulfur at various states of vulcanization for a number of rubber compounds have been studied. While the anticipated loss in unsaturation of the rubber hydrocarbonwas noted in stocks containing nonreinforcing fillers, no such loss in unsaturation could be detected in rubber compounds containing reinforcing channel blacks. It is suggested that this alteration in the mechanism of sulfur vulcanization may be mostly responsible for the physical characteristics of reinforced rubber stocks. It has not been possible to detect any effect of carbon black on the unsaturation value of natural rubber. Calculation indicates that, while such an effect would not be detected in the present case with the analytical method employed, such an effect, if present, should be detectable with carbon blacks of greater surface area than those employed in the present investigation.
EVERAL investigators (20)have established that sulfur
S
vulcanization of rubber involves chemical combination of sulfur and rubber hydrocarbon. A definite decrease in unsaturation of the rubber hydrocarbon as vulcanization progresses has been generally noted (S, 10, 18). While it has often been concluded that a double bond is saturated for each atomic equivalent of combined sulfur (10, 18), recent work by Brown and Hauser (3, 7 ) demonstrates that this conclusion cannot be applied in all cases. In certain compounds they found the loss in unsaturation with extent of vulcanization to be considerably less than anticipated on the above basis. Their results indicated that stocks reaching optimum cure with the least loss of unsaturation possessed the greatest tensile strength. Although a considerable amount of work has been done on this problem, we have not found a published account of simi-
lar investigations performed on stocks containing significant loadings of reinforcing fillers. Since, as pointed out below, the nature of the bonding between such fillers and the rubber molecule has not been clearly defined, one is not justified in applying previous results obtained on stocks containing no reinforcing fillers t o those bearing appreciable loadings of such substances. Accordingly one portion of the present investigation was concerned with determining the effect of various. fillers on the course of sulfur vulcanization, as judged from combined sulfur and unsaturatioii values. Aa pointed out by Gehman and Field (S), it is undoubtedly true that the black particle in a carbon-black-reinforced rubber stock is firmly attached to the rubber molecule. The nature of the bonding between the black and rubber has not been clearly defined. Some investigators (4,8, 14, 17) maintain that the association is physical and involves dcfinitc forces of adhesion or adsorption; othcrs ( I S ) have suggested formation of primary valence linkages with the rubber hydrocarbon. The opinion of the present autho& is that if such linkages are formed, the ethylenic bonds of the rubber molecule would probably beinvolved. If this latter view is correct, then a specific loss in unsaturation of the rubber hydrocarbon, due to the reinforcing filler, should occur. Thus the second objective of the present study was to determine whether it was possible by chemical means t o detect such a linkage. If measurable, this effect, together with the surface area determinations reported previously (15), would be particularly valuable in estimating the reinforcing value of various fillers.
Experimental Procedure The unsaturation of the rubber stocks was determined b y addition of iodine chloride. The procedure followed was essentially Kemp’s technique (9, 11) as modified by Blake and Bruce ( 9 ) : A 0.1-gram sample of stock was dissolved in boiling p-dichlorobenzene. This usually required from 2 to 3 hours. Solution became more difficult with well cured compounds. This was over
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